PROJECT SUMMARY The c-kit-positive (c-kit+) cells are the first putative population of cardiac stem cells (CSCs) identified in mammals, with self-renewing, clonogenic and multipotent activities in vitro. It also reported that adult cardiac c- kit+ cells are necessary and sufficient for myocardial regeneration. While these findings are encouraging, a recent lineage tracing study in mice indicated that cardiac resident c-kit+ cells have minimal potential to differentiate into cardiomyocytes in vivo. To ascertain the true identity of cardiac c-kit+ cells, we generated a series of new mouse models by targeting reporter genes (H2B-tdTomato, nuclear lacZ and H2B-GFP) and MerCreMer cassette into the start codon of c-kit in mice. With them, we first uncovered that c-kit in fact labels a subpopulation (~43%) of PECAM+ cardiac endothelial cells. After acute cardiac injury, the resident c-kit+ cells still retain their endothelial identity, and have little or no potential to become cardiomyocytes. However, disregard the low myogenic potential of cardiac resident c-kit+ cells during development and after cardiac injury, transplantation of exogenously expanded c-kit+ cells has been consistently shown to improve heart function and attenuate adverse left ventricular remodeling in both ischemic and non-ischemic cardiomyopathy. Thus, there must be unrecognized mechanisms underlying c-kit+ cell therapy. Given our new finding that c-kit actually labels a subpopulation of cardiac endothelial cells, we hypothesized transplanted c-kit+ cells repair the injured heart through their endothelial nature. c-kit+ cells may improve self-renew of the recipient heart by generating new coronary vasculature, or by releasing paracrine factors. Transplanted c-kit+ cells may also be reprogramed and gain multipotency after in vitro expansion. In this research program, we will use state-of-the- art mouse models and human c-kit+ cardiac endothelial cells to test these hypotheses with three Aims: Aim 1 will determine if the endothelial nature of c-kit+ cells contributes to heart repair; Aim 2 will determine if the transplanted c-kit+ endothelial cells promote self-renew of the recipient heart, and if they serve as progenitors to regenerate coronary vessels and/or cardiomyocytes; Aim 3 will determine if the paracrine factors from transplanted c-kit+ cells are essential for heart repair. This project will provide definitive answers to elucidate ultimate mechanisms by which c-kit+ cells promote heart repair.